The long term objective of this research proposal is to better understand hepatic nutrient sensing and its effects on cellular metabolism and insulin sensitivity. Hepatocytes exposed to a hyperglycemic environment in vitro and in vivo show evidence of lipid accumulation, inflammation, and insulin resistance independent of hormonal factors. The mechanism by which this occurs is not completely understood, despite the fact that it may play a key role in the pathological response to chronic hyperglycemia associated with type 2 diabetes. Two proteins that stand out as candidates for the study of this process are SIRT1 and AMPK. SIRT1 belongs to the sirtuin family of redox-sensitive deacetylases that can be activated by fasting and are thought to account for the increased longevity associated with caloric restriction. AMP-activated protein kinase (AMPK) is a fuel-sensing molecule that increases ATP production and decreases ATP consumption during periods of fasting or exercise. Decreases in AMPK activity have been described in various animal models with insulin resistance, and increasing glucose concentration has been shown to decrease the abundance of both AMPK and SIRT1 in cultured hepatocytes. Conversely, activators of both SIRT1 and AMPK have been shown to increase insulin sensitivity in vitro and in vivo. The seemingly parallel activity of these molecules and their inherent similarities in sensing fuel supply and regulating cellular metabolism suggests that substantial cross-talk may exist between them. Accordingly, the goal of this research proposal is to test the hypothesis that SIRT1 regulates AMPK in response to fluctuations in hepatic glucose concentration and/or redox state. Cultured hepatoma cells (HepG2) will be used in Aim 1 to confirm previous observations regarding parallel SIRT1 and AMPK activity, to assess their relation to changes in cytosolic redox state, and to determine whether AMPK activation in certain settings is dependent on SIRT1 activity. Aim 2 will explore the hypothesis that SIRT1 activates AMPK via modulation of the AMPK kinase LKB1 by assessing LKB1 deacetylation and activation in HepG2 cells incubated with known SIRT1 and AMPK activators. Aim 3 will examine whether prevention of insulin resistance by AMPK activation is associated with and/or requires activation of SIRT1 or LKB1. Lastly, Aim 4 will explore whether the in vitro observations are confirmed in vivo by studying the effect of glucose infusions on SIRT1, LKB1, and AMPK activity in rat liver. This research will further the understanding of how high blood glucose may adversely affect liver function in patients with diabetes. It could also lead to new therapeutic strategies to prevent diabetic complications.